Forkhead box O3 promotes cell proliferation and inhibits myotube differentiation in chicken myoblast cells

1. In the poultry industry, growth performance is important due to its effects on economic value. Much effort has been put forth to achieve introgression of specific genes and DNA markers related to muscle proliferation and differentiation in selective breeding approaches.

2. This study investigated the biological functions of the gene Forkhead box O3 (FOXO3) during myogenic differentiation in chicken myoblast cells. FOXO3 was downregulated in primary chicken myoblast (pCM) cells by the piggyBac transposon-mediated microRNA (miRNA) knock-down (KD) system.

3. The pCM cells that were stably integrated into the FOXO3 KD expression vector showed significant downregulation of FOXO3 protein and mRNA levels. Expression levels of paired box protein Pax7 (Pax7) and target genes such as CCAAT/enhancer binding protein beta and serum response element decreased in FOXO3 KD pCM cells. In addition, in the undifferentiated myoblast stage, there were no significant differences in cell morphology; however, proliferation rate in FOXO3 KD pCM cells was significantly lower during d 4 and 5 of in vitro culture. By contrast, when myotube differentiation was induced, FOXO3 KD pCM cells exhibited rapid initiation of myotube formation, higher expression of myogenin and desmin as myogenic indicators and a further differentiated phenotype than observed in regular pCM cells.

4. These results demonstrated that FOXO3 promotes cell proliferation and inhibits myotube differentiation in chicken myoblast cells. Therefore, the regulation of FOXO3 could be applied to improve muscle differentiation in commercial poultry.     

Reference gene selection for reverse transcription quantitative polymerase chain reaction in chicken hypothalamus under different feeding status

This study was designed to investigate the stability of 10 candidate reference genes, namely ACTB, B2M, GAPDH, HMBS, LBR, POLR2B, RN18S, RPS17, TBP, and YWHAZ for the normalization of gene expression data obtained by quantitative real‐time polymerase chain reaction (qPCR) in studies related to feed intake of chicken. Samples were isolated from hypothalamus under three different nutritional status (ad libitum, fasted for 24 hr, fasted for 24 hr then refed for 2 hr). Five different algorithms were applied for the analysis of reference gene stability: BestKeeper, geNorm, NormFinder, the comparative ΔCt method, and a novel approach using multivariate linear mixed‐effects modelling for stable reference gene selection. TBP and POLR2B were identified as the two most suitable and B2M and RN18S as the two least stable reference genes for normalization. Despite our review, the current literature showing that RN18S is one of the most commonly used reference gene in chicken gene expression studies, its applicability for normalization should be evaluated before each qPCR experiment.     

Expression of Mitochondria‐Associated Genes (PPARGC1A,NRF‐1, BCL‐2 and BAX) in Follicular Development and Atresia of Goat Ovaries

Most follicles undergo atresia during the developmental process. Follicular atresia is predominantly regulated by apoptosis of granulosa cells, but the mechanism underlying apoptosis via the mitochondria‐dependent apoptotic pathway is unclear. We aimed to investigate whether the mitochondria‐associated genes peroxisome proliferator‐activated receptor‐gamma, coactivator1‐alpha (PPARGC1A), nuclear respiratory factor‐1 (NRF‐1), B‐cell CLL/lymphoma 2 (BCL‐2) and BCL2‐associated X protein (BAX) played a role in follicular atresia through this pathway. The four mitochondria‐associated proteins (PGC‐1α, which are encoded by the PPARGC1A gene, NRF‐1, BCL‐2 and BAX) mainly expressed in granulosa cells. The mRNA and protein levels of PPARGC1A/PGC‐1α and NRF‐1 in granulosa cells increased with the follicular development. These results showed that these genes may play a role in the regulation of the follicular development. In addition, compared with healthy follicles, the granulosa cell in atretic follicles had a reduced expression of NRF‐1, increased BAX expression and increased ratio of BAX to BCL‐2 expression. These results suggested that changes of the mitochondria‐associated gene expression patterns in granulosa cells may lead to follicular atresia during goat follicle development.     

Ruminal epithelial insulin‐like growth factor‐binding proteins 2, 3, and 6 are associated with epithelial cell proliferation

The aim of this study was to identify factors that regulate ruminal epithelial insulin‐like growth factor‐binding protein (IGFBP) expression and determine its role in rumen epithelial cell proliferation. Primary bovine rumen epithelial cells (BREC) were incubated with short‐chain fatty acids (SCFAs) at pH 7.4 or 5.6, lactate, lipopolysaccharide (LPS), insulin‐like growth factor‐I (IGF‐I), ‐II (IGF‐II), or recombinant bovine IGFBP2 (rbIGFBP2). The mRNA expression levels of IGFBP in BREC were analyzed using quantitative real‐time polymerase chain reaction (qRT‐PCR). The proliferation rate of BREC was analyzed using a WST‐1 assay. IGFBP2 gene expression tended to be lower with SCFA treatment (p < .1), and IGFBP6 gene expression was significantly lower with SCFA treatment (p < .05). IGFBP3 and IGFBP6 gene expression tended to be higher with d ‐Lactate treatment (p < .1). IGFBP3 gene expression was significantly higher (p < .05) with LPS treatment. BREC treated with IGF‐I grew more rapidly than vehicle control‐treated cells (p < .01); however, recombinant bovine rbIGFBP2 inhibited IGF‐I‐induced proliferation. IGF‐II and/or rbIGFBP2 did not affect BREC proliferation. Taken together, SCFA treatment decreased IGFBP2 and IGFBP6 expression in rumen epithelial cells, and lower expression of these IGFBP might promote rumen epithelial cell proliferation by facilitating IGF‐I.     

Effect of chicken leptin recptor short hairpin RNA on expression of JAK2, STAT3, SOCS3 and CPT1 genes in chicken preadipocytes

In this study, we detect depressive effect on leptin receptor (LEPR) by LEPR‐specific short hairpin RNA (shRNA) expression plasmids in chicken preadipocytes, and effect on messenger RNA (mRNA) expression levels of genes related to signal transduction, including JAK2, STAT3, SOCS3 as well as CPT1, which is associated with fatty acid metabolism. shRNA expression vectors targeting LEPR were constructed and transfected into chicken preadipocytes. The transfection efficiency was evaluated by fluorescence microscopy. Real‐time PCR was used to detect its effect on mRNA expression levels of JAK2, STAT3, SOCS3 and CPT1. Results showed that LEPR mRNA was knocked down by 99% (P < 0.01) after transfection for 72 h. In the knockdown preadipocytes, the mRNA levels of JAK2 and CPT1 were down‐regulated by 47.56% (P < 0.01) and 42.26% (P < 0.05), respectively; while expression of STAT3 and SOCS3 increased 7.72‐fold (P < 0.01), 1.71‐fold (P < 0.01), respectively. It is concluded that knockdown of LEPR influences mRNA expression of its down‐stream genes, suggesting that chicken LEPR play a certain role in regulating genes in the complicated gene network of preadipocytes.     

Sequence of the domestic duck (Anas platyrhynchos) growth hormone-encoding gene and genetic variation in the promoter region

The duck growth hormone encoding gene and its promoter region were amplified by polymerase chain reaction (PCR). A total of 5.25 kb were cloned and sequenced. Duck growth hormone (GH) consists of five exons and four introns and is structurally similar to mammalian and chicken GH gene. Although the distal region of duck GH promoter showed no similarity to chicken and turkey promoters, the proximal region of the promoter contained two putative Pit‐1 binding sequences, and showed similarity to chicken and turkey GH promoters. Genetic variation was detected at five positions of the promoter region. The results of this study indicate that the expression of duck GH is likely regulated in a similar manner to that of chicken GH via enhancer‐type cis‐acting elements and the presence of genetic variation in the duck GH gene may be applicable to marker‐assisted selection.     

Cytogenetic mapping of 11 functional genes to chicken chromosomes by fluorescence in situ hybridization

With chicken bacterial artificial chromosome (BAC) DNA as probes, 11 non‐assigned functional genes were localized to chicken chromosomes 1 or 2 by fluorescence in situ hybridization (FISH). The 11 genes and their chromosomal positions are as follows: ALVEB5, 1p26‐24; ACO2, 1p16‐14; HSP108, 1p14‐13; CD4, 1q11; HSD3B; 1q11; SOD1, 1q14‐21; LAMP1, 1q24‐31; P2Y5, 1q35‐36; EN2, 2p31‐24; NPY, 2p14‐13 and CA2, 2q31‐32. Metaphase chromosome spreads used for hybridization were prepared from embryonic chicken fibroblast cultures. The gene position was identified according to the international standardized G‐banded karyotype of chicken by measuring the relative fractional length from the telomere of the p‐arm to the hybridization signal (FLpter). The 11 genes mapped newly will enrich the cytogenetic map and serve as additional anchor markers for integrating the cytogenetic map with the genetic map of chicken.     

Does the tumour microenvironment alter tumorigenesis and clinical response in transmissible venereal tumour in dogs?

The canine transmissible venereal tumour (CTVT) is a transmissible cancer that is spread naturally between dogs, with the ability to develop and evade the immune system, despite strict immune surveillance of the host. Furthermore, molecular signalling between cells of the immune system and the tumour microenvironment appear to influence the behaviour and development of the tumour. Thus, this study aimed to quantify the expression of genes related to the immune system such as IL‐6, IFN‐γ, and TGF‐β, as well as angiogenic factors (VEGF, CXCR4), in CTVT cells in vivo and in vitro (primary culture), correlating with the clinical response of the animals treated with vincristine. As expected, the most prevalent subtype was plasmacytoid cells, although lymphocytic cells were also found, indicating the possibility of polyclonality. When we compared the gene expressions of IFN‐γ and IL‐6, we mostly found low expression, concluding that MHC expression was probably not occurring in tumour cells, and no activation of immune cells to eliminate the tumour. The TGF‐β gene was normal in the majority of animals but demonstrated decreased expression in vincristine resistant animals, leading to the hypothesis that the concentration of tumour‐derived TGF‐β was affecting and even suppressing the real TGF‐β expression, favouring tumour proliferation and progression in these cases. VEGF expression was extremely high, demonstrating its angiogenic role in tumour growth, while CXCR4 was decreased, possibly because of CTVT’s low metastatic potential. Thus, we concluded that the tumour microenvironment, together with the immune system of the host, influences CTVT, presumably altering its tumorigenesis and the animal’s clinical response to treatment.     

Effect of retinoic acid on gene expression profiles of bovine intramuscular preadipocytes during adipogenesis

To investigate genes involved in intramuscular adipogenesis in ruminants, 16 genes with dramatic variable expression were selected. These were selected from the differentiation‐ and proliferation‐phase libraries of our previous serial analysis of gene expression (SAGE) studies of a clonal bovine intramuscular preadipocyte (BIP) cell line. We harvested the BIP cells over 12 days after adipogenic stimulation with all‐trans retinoic acid (ATRA). Quantitative real‐time PCR confirmed the earlier SAGE study results of the expression patterns of 15 of the genes. On day 6, TG accumulation increased significantly in the BIP cells but was completely inhibited in the 3T3‐L1 cells (the monogastric reference). ATRA enhanced expression levels of six genes whereas it suppressed expression of eight genes on day 3 of adipogenesis in the BIP cells. Forty‐eight hours after transfection, the messenger RNA expression level of the adipose differentiation‐related protein (ADFP), encoded by one of the upregulated genes, in the ADFP small interference RNA (siRNA)‐transfected cells was 3.5% of that in negative control‐transfected cells. Also, 6 days after induction the TG level in the ADFP siRNA‐transfected cells was 21.8% lower than that in negative control‐transfected cells. This analysis of gene expression profiles after ATRA treatment will contribute to our understanding of the molecular mechanisms involved in bovine intramuscular adipogenesis.     

MicroRNA‐145 regulates immune cytokines via targeting FSCN1 in Staphylococcus aureus‐induced mastitis in dairy cows

Dairy cow mastitis is a detrimental factor in milk quality and food safety. Mastitis generally refers to inflammation caused by infection by pathogenic microorganisms. Our studies in recent years have revealed the role of miRNA regulation in Staphylococcus aureus‐induced mastitis. In the present study, we overexpressed and suppressed miR‐145 to investigate the function of miR‐145 in Mac‐T cells. Flow cytometry, ELISA and EdU staining were used to detect changes in the secretion of several Mac‐T cytokines and in cell proliferation. We found that overexpression of miR‐145 in Mac‐T cells significantly reduced the secretion of IL‐12 and TNF‐α, but increased the secretion of IFN‐γ; the proliferation of bovine mammary epithelial cells was also inhibited. Using quantitative real‐time PCR (qRT‐PCR), Western blotting and luciferase multiplex verification techniques, we found that miR‐145 targeted and regulated FSCN1. Knock‐down of FSCN1 significantly increased the secretion of IL‐12, while the secretion of TNF‐α was significantly downregulated in Mac‐T cells. Upon S. aureus infection of mammary gland tissue, the body initiated inflammatory responses; Bta‐miR‐145 expression was downregulated, which reduced the inhibitory effect on the FSCN1 gene; and upregulation of FSCN1 expression promoted mammary epithelial cell proliferation to allow the recovery of damaged tissue. The results of the present study will aid in understanding the immune mechanism opposing S. aureus infection in dairy cows and will provide a laboratory research basis for the prevention and treatment of mastitis.     

Molecular and Statistical Analysis of Campylobacter spp. and Antimicrobial‐Resistant Campylobacter Carriage in Wildlife and Livestock from Ontario Farms

The objectives of this study were to (i) compare the carriage of Campylobacter and antimicrobial‐resistant Campylobacter among livestock and mammalian wildlife on Ontario farms, and (ii) investigate the potential sharing of Campylobacter subtypes between livestock and wildlife. Using data collected from a cross‐sectional study of 25 farms in 2010, we assessed associations, using mixed logistic regression models, between Campylobacter and antimicrobial‐resistant Campylobacter carriage and the following explanatory variables: animal species (beef, dairy, swine, raccoon, other), farm type (swine, beef, dairy), type of sample (livestock or wildlife) and Campylobacter species (jejuni, coli, other). Models included a random effect to account for clustering by farm where samples were collected. Samples were subtyped using a Campylobacter‐specific 40 gene comparative fingerprinting assay. A total of 92 livestock and 107 wildlife faecal samples were collected, and 72% and 27% tested positive for Campylobacter, respectively. Pooled faecal samples from livestock were significantly more likely to test positive for Campylobacter than wildlife samples. Relative to dairy cattle, pig samples were at significantly increased odds of testing positive for Campylobacter. The odds of isolating Campylobacter jejuni from beef cattle samples were significantly greater compared to dairy cattle and raccoon samples. Fifty unique subtypes of Campylobacter were identified, and only one subtype was found in both wildlife and livestock samples. Livestock Campylobacter isolates were significantly more likely to exhibit antimicrobial resistance (AMR) compared to wildlife Campylobacter isolates. Campylobacter jejuni was more likely to exhibit AMR when compared to C. coli. However, C. jejuni isolates were only resistant to tetracycline, and C.  coli isolates exhibited multidrug resistance patterns. Based on differences in prevalence of Campylobacter spp. and resistant Campylobacter between livestock and wildlife samples, and the lack of similarity in molecular subtypes and AMR patterns, we concluded that the sharing of Campylobacter species between livestock and mammalian wildlife was uncommon.     

Prokaryotic expression of chicken interferon‐γ fusion protein and its effect on expression of poultry heat shock protein 70 under heat stress

Interferons have attracted considerable attention due to their vital roles in the host immune response and low induction of antibiotic resistance. In this study, total RNA was extracted from spleen cells of chicken embryos inoculated with Newcastle disease vaccine, and the full‐length chicken interferon‐γ (ChIFN‐γ ) gene was amplified by RT‐PCR. The full complementary DNA sequence of the ChIFN‐γ gene was 495 bp long and was cloned into the prokaryotic expression vector pProEX?HT_b. The plasmid was transformed into Escherichia coli DH5α and the expression of ChIFN‐γ was induced by isopropyl β‐D‐1‐thiogalactopyranoside. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis and Western blot results showed the expressed fusion protein had a molecular weight of approximately 18 kDa and was recognized by an anti‐His mAb. Moreover, ChIFN‐γ was found to demonstrate anti‐viral activity in vitro . To test the in vivo function of ChIFN‐γ in broilers under heat stress, a total of 100 broilers were randomly assigned to either a control group or a treated group, in which they were hypodermically injected with recombinant ChIFN‐γ. Results demonstrated ChIFN‐γ affects the messenger RNA expression levels of heat shock protein 70 (HSP70) in the heart and lung tissues, and decreases the concentration of HSP70 in serum. Therefore, we conclude recombinant ChIFN‐γ can reduce heat stress to some extent in vivo .     

Gossypol inhibits LH‐induced steroidogenesis in bovine theca cells

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0‐25 μg/mL) for 24 h. Gossypol inhibited LH‐stimulated theca cell A4 and P4 production in a dose‐dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down‐regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down‐regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.     

Aspartame supplementation in starter accelerates small intestinal epithelial cell cycle and stimulates secretion of glucagon‐like peptide‐2 in pre‐weaned lambs

The objective of this study was to test the hypothesis that aspartame supplementation in starter diet accelerates small intestinal cell cycle by stimulating secretion and expression of glucagon‐like peptide ?2 (GLP‐2) in pre‐weaned lambs using animal and cell culture experiments. In vivo, twelve 14‐day‐old lambs were selected and allocated randomly to two groups; one was treated with plain starter diet (Con, n = 6) and the other was treated with starter supplemented with 200 mg of aspartame/kg starter (APM, n = 6). Results showed that the lambs received APM treatment for 35 d had higher (p < .05) GLP‐2 concentration in the plasma and greater jejunum weight/live body weight (BW) and jejunal crypt depth. Furthermore, APM treatment significantly upregulated (p < .05) the mRNA expression of cyclin D1 in duodenum; and cyclin A2, cyclin D1, cyclin‐dependent kinases 6 (CDK6) in jejunum; and cyclin A2, cyclin D1, CDK4 in ileum. Moreover, APM treatment increased (p < .05) the mRNA expression of glucagon (GCG), insulin‐like growth factor 1 (IGF‐1) in the jejunum and ileum and mRNA expression of GLP‐2 receptor (GLP‐2R) in the jejunum. In vitro, when jejunal cells were treated with GLP‐2 for 2 hr, the 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide (MTT) OD, IGF‐1 concentration, and the mRNA expression of IGF‐1, cyclin D1 and CDK6 were increased (p < .05). Furthermore, IGF‐1 receptor (IGF‐1R) inhibitor decreased (p < .05) the mRNA expression of IGF‐1, cyclin A2, cyclin D1 and CDK6 in GLP‐2 treatment jejunal cells. These results suggest that aspartame supplementation in starter accelerates small intestinal cell cycle that may, in part, be related to stimulate secretion and expression of GLP‐2 in pre‐weaning lambs. Furthermore, GLP‐2 can indirectly promote the proliferation of jejunal cells mainly through the IGF‐1 pathway. These findings provide new insights into nutritional interventions that promote the development of small intestines in young ruminants.     

Adiponectin‐impaired adipocyte differentiation negatively regulates fat deposition in chicken

Adiponectin is a protein hormone secreted exclusively by adipocytes that plays an important role in the modulation of glucose and lipid metabolism. To investigate the effect of adiponectin on lipid metabolism in chicken, rosiglitazone (agonist of adiponectin) and dexamethasone (inhibitor of adiponectin) were used to treat 23‐day‐old broilers in vivo. To verify the functionality of adiponectin on fat deposition, chicken pre‐adipocytes were cultured in the medium containing 10 μg/ml adiponectin. Serum adiponectin and lipids and fat distribution were analysed. Oil Red O staining was used to determine lipid deposition in adipocytes. The expression levels of adiponectin, adiponectin receptors (AdipoR) and lipid metabolism–related genes in different tissues and pre‐adipocytes were measured using real‐time PCR, and the abundance of lipid metabolism–related proteins was measured by Western blot. Rosiglitazone increased serum adiponectin concentration and the expression levels of adiponectin and adiponectin receptor 1 (AdipoR1) in tissues and significantly decreased levels of serum lipids and fat deposition. Rosiglitazone significantly increased the expression levels of adipose triglyceride lipase (ATGL) and AdipoR1 and decreased the expression levels of fatty acid synthase (FAS). Dexamethasone had the converse effects compared with rosiglitazone. Oil red O staining results showed a marked decrease in fat deposition in cells treated with adiponectin. In adipocytes, adiponectin could decrease the expression levels of CCAAT/enhancer‐binding protein α (C/EBPα) and FAS and increased the expression levels of ATGL and AdipoR1. These results indicate that adiponectin has a remarkable effect on impairment of adipocyte differentiation, which contributes to the negative regulation of fat deposition in chicken.